This proposal will examine erythroid stimulators which may be involved in the regulation of normal erythropoiesis. Burst promoting activity (BPA), erythroid colony forming unit activity (CPA) and proerythroblast stimulating activity (PSA) will be examined. BPA has been detected in certain spleen cell conditioned media and enhances the proliferation of the erythroid burst forming unit (BFU-E). The same conditioned media also contains CPA which stimulates the more mature erythroid colony forming units (CFU-E). Mouse serum contains PSA which is a more potent stimulator of proerythroblast proliferation than erythropoietin (Ep). These stimulators will be concentrated and purified by a variety of biochemical procedures including ammonium sulphate precipitation, Sephadex, Sepharose and anion exchange chromatography, preparative analytical isoelectric focussing and preparative analytical polyacrylamide electrophoresis. Once sufficiently pure preparations are available antibodies will be raised against them. An analysis of their molecular weight, electrophoretic properties, immunologic cross reactivities as well as their functional properties will allow the assessment of the number of stimulators enhancing erythropoietic activity. Initial studies will determine the effect of each of the stimulators on BFU-E, CFU-E and proerythroblasts by adding them at varying concentrations to appropriate in vitro culture system. The addition of specific antibody will confirm that enhanced colony or cell growth is indeed due to the stimulator. Once the optimum concentration of stimulator is known the effect of adding them in combination will be examined. In each of the studies the minimum requirement of Ep essential for maximal colony growth in the presence of the stimulators will be determined. It is our hypothesis that the addition of relatively pure more concentrated stimulators of erythropoiesis, either alone or in combination, will allow in vitro erythroid colony or cell growth at far lower (more physiological concentrations) of Ep and hence allow a better understanding of the normal multifactorial nature of the regulation of erythropoiesis.